The present invention is related to syntheses of novel isodethiaazacephems and isodethiaazacephams, and their use as an potent antibacterial agent, and in particular isodethiaazacephems and isodethiaazacephams having an effective leaving group which can undergo an enzyme-initiated elimination process, so that the antibacterial activity thereof can be enhanced.
xcex2-Lactam antibiotics exert certain biological activity by acylating serine residues of transpeptidases so that the cross-linking of peptidoglycans does not take place [Waxman, D. J.; Strominger, J. L. Sequence of Active Site Peptides from the Penicillin-sensitive D-Alanine Carboxypeptidase of Bacillus Subtilis. J. Biol. Chem. 1980, 255, 3964-3976; FrËre, J. M.; Nguyen-DistËche, M.; Coyette, J.; Joris, B. Mode of Action: Interaction with the Penicillin Binding Proteins. In The Chemistry of xcex2-Lactams; Page, M. I., Ed.; Blackie Academic and Professional: New York, 1992; pp 148-197]. As shown in Scheme 1, ring opening of the xcex2-lactam nucleus would occur when cephalosporins (1) react with enzymes responsible for the cell wall synthesis of bacteria. Consequently, the substituent at the C-3xe2x80x2 position is liberated [Boyd, D. B. Elucidating the Leaving Group Effect in the xcex2-Lactam Ring Opening Mechanism of Cephalosporins. J. Org. Chem. 1985, 50, 886-888; Boyd, D. B.; Lunn, W. H. W. Electronic Structures of Cephalosporins and Penicillins. 9. Departure of a Leaving Group in Cephalosporins. J. Med. Chem. 1979, 22, 778-784; Faraci, W. S.; Pratt, R. F. Elimination of a Good Leaving Group from the 3xe2x80x2-Position of a Cephalosporin Need Not Be Concerted with xcex2-Lactam Ring Opening. J. Am. Chem. Soc. 1984, 106, 1489-1490; Page, M. L.; Proctor, P. Mechanism of xcex2-Lactam Opening in Cephalosporins. J. Am. Chem. Soc. 1984, 106, 3820-3825; Grabowski, E. J. J.; Douglas, A. W.; Smith, G. B. Ammonolysis of Cephalosporins: 13C NMR Characterization of the Intermediates from xcex2-Lactam Ring Cleavage Prior to Loss of the 3xe2x80x2-Group. J. Am. Chem. Soc. 1985, 107, 267-268]. When the eliminated species possesses excellent leaving ability, cephalosporins (1) may exhibit profound antibacterial activity. 
Accordingly, we designed and synthesized unprecedented isodethiaazacephems having the following formula (I): 
wherein RI is hydrogen or xe2x80x94SO2RIII, preferably xe2x80x94SO2RIII;
RII is xe2x80x94CO2RIV or xe2x80x94SO2RIII, preferably xe2x80x94CO2RIV; and
RV is a substituted acetamido radical;
wherein RIII is a hydrogen, C1-C6 alkyl, aralkyl having a total carbon number of 7-12, aryl, or a halogenated C1-C6 alkyl; and RIV is a hydrogen, C1-C6 alkyl, aralkyl having a total carbon number of 7-12 or aryl.
We believe that the sulfone moiety, xe2x80x94SO2RIII, at the Oxe2x80x943xe2x80x2 position of (I) could act as an effective leaving groups, and thus further enhance the antibacterial activity in comparison with that of the parent 3-(hydroxy)isodethiaazacephem (RI is hydrogen).
Recognizing the feasibility of 1,4-elimination in xcex2-lactam antibiotics as shown in Scheme 1, we also synthesized novel isodethiaazacephams having the following formula (II): 
wherein RIII, RIV and RV are defined as above.
The newly designed compound (II) bears a leaving group at the C-4 position; the [1,2]-elimination process could also be initiated by bacterial enzymes (See Scheme 2).
Preferably, RIII in the formula (I) is C1-C6 alkyl or halogenated C1-C6 alkyl, more preferably xe2x80x94CH3 or xe2x80x94CF3, and most preferably, xe2x80x94CF3.
Preferably, RIII in the formula (II) is C1-C6 alkyl, and more preferably xe2x80x94CH3.
Preferably, RIV in the formulas (I) and (II) is hydrogen.
Preferably, RV in the formulas (I) and (II) is phenylacetamido.
The present invention provides new classes of xcex2-lactams (i.e. isodethiaazacephems and isodethiaazacephams) possessing notable antibacterial activity. In the following preferred embodiments of the present invention, we synthesized compounds 3-17, in which compounds 3, 4, 9-12 and 17 are isodethiaazacephem derivatives, and compounds 5 and 16 are isodethiaazacepham derivatives. The reaction routs for the synthesis of these xcex2-lactams are illustrated in Schemes 3-5. Compounds (xc2x1)xe2x88x923, (xc2x1)xe2x88x924 and (xc2x1)xe2x88x925 shown as follows are prominent examples among the general structures (I) and (II): 
Reagents: (a) NaN3, DMF, r.t. (90%); (b) CF3SO2Cl, Et3N, CH2Cl2, 0xc2x0 C.xe2x86x92r.t. (90%); (c) Pd/C, H2, EtOAc, r.t., 7xe2x86x929 (94%); (d) Pd/C, H2, EtOAc, r.t., 8xe2x86x929 (87%); (e) PdCl2, H2, EtOH, r.t. (50%); (f) MeSO2Cl, pyridine, CH2Cl2, 15xc2x0 C., 9xe2x86x9211 (45%); (g) CF3SO2Cl, pyridine, CH2Cl2, 15xc2x0 C., 9xe2x86x9212 (30%)+13 (10%); (h) PdCl2, H2, EtOH, r.t., 11xe2x86x923 (35%), 12xe2x86x924 (30%). 
Reagents: (a) H2S, Et3N, CH2Cl2, r.t. (55%), 8xe2x86x929 (15%)+14 (40%). 
Reagents: (a) MeSO2Cl, Et3N, CH2Cl2, 0xc2x0 C. (85%); (b) Pd/C, H2, EtOAc, r.t. (90%); (c) PdCl2, H2, EtOH, r.t., 16xe2x86x925 (20%)+17 (50%).
For the synthesis of isodethiaazacephems (xc2x1)xe2x88x923 and (xc2x1)xe2x88x9210, we treated racemic xcex2-lactam mesylate 6 with NaN3 in DMF at room temperature to give azido xcex2-lactam 7 in 90% yield (Scheme 3) [Hakimelahi, G. H.; Just, G.; Ugolini, A. The Synthesis of an O-2-Isooxacephem. Helv. Chim. Acta 1982, 65, 1368-1373]. Catalytic hydrogenation of 7 by use of Pd/C and H2 (30-35 psi) in EtOAc at room temperature resulted in the reduction of the azide moiety and spontaneous formation of isodethiaazacephem 9 in 94% yield. Debenzylation of 9 by use of PdCl2 and H2 (60 psi) in EtOH produced the target isodethiaazacephem (xc2x1)xe2x88x9210 in 50% yield.
An alternative way to obtain 9 from azido xcex2-lactam 7 involved two steps. Chlorination of 7 with CF3SO2Cl in Et3N and CH2Cl2 produced chloride 8 in 90% yield [Hakimelahi, G. H.; Tsay, S.-C.; Ramezani, Z.; Hwu, J. R. Syntheses of New Isocephems and Isodethiaoxacephems as Antimicrobial Agents. Helv. Chim. Acta 1996, 79, 813-819]. Consequent reduction of 8 by use of Pd/C and H2 (30-35 psi) in EtOAc gave the desired compound 9 in 87% yield (Scheme 3). On the other hand, reaction of 8 with H2S in Et3N and CH2Cl2 produced a mixture of isodethiaazacephem 9 (15%) and isodethiaazapenam 14 (40%) as shown in Scheme 4.
We attached a sulfonyl group onto the cephem nucleus of 9 by mesylation with MeSO2Cl in pyridine and CH2Cl2 to give the 3-mesyloxy xcex2-lactam 11 in 45% yield (Scheme 3). It was then hydrogenated with PdCl2 in EtOH at 60 psi of H2 to produce the desired isodethiaazacephem (xc2x1)xe2x88x923 in 35% yield. Moreover, we treated xcex2-lactam 9 with CF3SO2Cl in pyridine and CH2Cl2 to afford a 3:1 mixture of trifluoromethanesulfonates 12 and 13 in 40% overall yield. Catalytic reduction of 12 with PdCl2 in EtOH at 60 psi of H2 gave the target isodethiaazacephem (xc2x1)xe2x88x924 in 30% yield.
For the synthesis of isodethiaazacepham (xc2x1)xe2x88x925 bearing a methylsulfonyl group at the C-4 position, we treated racemic azido xcex2-lactam 7 with MeSO2Cl in Et3N and CH2Cl2 (Scheme 5). Sulfone 15, generated in 85% yield, was treated with H2 (30-35 psi) and Pd/C in EtOAc to give bicyclic xcex2-lactam 16 in 90% yield through sequential reduction and lactamization. Upon further reduction with H2 at 60 psi in the presence of PdCl2 and EtOH, compound 16 was converted to a mixture of the desired 4-substituted isodethiaazacepham (xc2x1)xe2x88x925 in 20% yield and the decarboxylated product (xc2x1)xe2x88x9217 in 50% yield.
We found that the solubility in water was 21 and 27 mg/mL for isodethiaazacephems (xc2x1)xe2x88x923 and (xc2x1)xe2x88x924, respectively; they were stable at physiological pH for six and four days, respectively. At pH 1.0, the xcex2-lactam rings in (xc2x1)xe2x88x923 and (xc2x1)xe2x88x924 survived for xcx9c4 and xcx9c2 h, respectively; yet at pH 12, they were destructed within 5-10 min. On the other hand, 3-(hydroxy)isodethiaazacephem (xc2x1)xe2x88x9210 was highly soluble (35 mg/mL) in a phosphate buffer (0.10 M, pH 6.8) and was stable at least for two months.
Isodethiaazacepham (xc2x1)xe2x88x925, highly soluble (32 mg/mL) in a phosphate buffer (0.10 M, pH 6.8), underwent decarboxylation gradually to give (xc2x1)xe2x88x9217 at room temperature within six days (Scheme 6). In a basic solution with pH 12, the decarboxylation also occurred to (xc2x1)xe2x88x925 within 20 min; in an acidic solution with pH 1.0, the xcex2-lactam ring in (xc2x1)xe2x88x925 was destructed within 3 h. The xcex2-lactam ring in (xc2x1)xe2x88x9217, however, was destructed at pH 1.0 within two days.
In another series of assays, we dissolved isodethiaazacephems (xc2x1)xe2x88x923 and (xc2x1)xe2x88x924 as well as isodethiaazacepham (xc2x1)xe2x88x925 in distilled water (5.0 mg/mL). The pH value of the aqueous solutions was kept initially about 4.0 for (xc2x1)xe2x88x923 and (xc2x1)xe2x88x924, and about 2.0 for (xc2x1)xe2x88x925. The pH values of the aqueous solutions of (xc2x1)xe2x88x923 and (xc2x1)xe2x88x924 changed to xcx9c1 within three and two days, respectively. We found that the change in pH was accompanied by the destruction of the xcex2-lactam rings in (xc2x1)xe2x88x923 and (xc2x1)xe2x88x924, as detected by IR spectroscopy, as well as the liberation of MeSO2H and CF3SO2H, respectively. On the other hand, the pH value of an aqueous solution of (xc2x1)xe2x88x925 changed from 2.0 to 6.0 within 5-6 days. This change was accompanied by the gradual production of (xc2x1)xe2x88x9217 through a decarboxylation process.
We tested the antibacterial activity of the synthesized xcex2-lactams (xc2x1)xe2x88x923, (xc2x1)xe2x88x924, (xc2x1)xe2x88x925, (xc2x1)xe2x88x9210, (xc2x1)xe2x88x9211, (xc2x1)xe2x88x9212, and (xc2x1)xe2x88x9217, as well as the reference compounds cefotaxime [Muhtadi, F. J.; Hassan, M. M. A. In Analytical Profiles of Drug Substances; Florey, K., Ed.; Academic: New York, 1982; Vol. 11, pp 139-168; Wise, R.; Rollason, T.; Logan, M.; Andrews, J. M.; Bedford, K. A. HR 756, A Highly Active Cephalosporin: Comparison with Cefazolin and Carbenicillin. Antimicrob. Agents Chemother. 1978, 14, 807-811] penicillin G [Morris, J. J.; Page, M. I. Intra- and Intermolecular Catalysis in the Aminolysis of Benzylpenicillin. J. Chem. Soc., Perkin Trans 2 1980, 212-219], and 7-(xcex2-phenylacetamido)-3xe2x80x2-desacetoxycephalosporanic acid (xc2x1)xe2x88x922 [Page, M. L.; Proctor, P. Mechanism of xcex2-Lactam Opening in Cephalosporins. J. Am. Chem. Soc. 1984, 106, 3820-3825] in vitro against five pathogenic microorganisms. The doses used were as high as 128 xcexcg/mL. The results are summarized in Table 1.
Results from biological tests reveal promising antimicrobial activities for the enol sulfonate xcex2-lactams (xc2x1)xe2x88x923 and (xc2x1)xe2x88x924. In comparison with the parent enol xcex2-lactam (xc2x1)xe2x88x9210, the enol sulfonate xcex2-lactams (xc2x1)xe2x88x923 and (xc2x1)xe2x88x924 exhibite much higher activity. Therefore, the antibacterial activity of (xc2x1)xe2x88x923 and (xc2x1)xe2x88x924 is enhanced substantially by possessing a potential leaving group at the O-3xe2x80x2 position.
Moreover, the trifluoromethanesulfone unit in (xc2x1)xe2x88x924 is a better leaving group than the methanesulfone unit in (xc2x1)xe2x88x923. Thus, antibacterial activity is more potent for (xc2x1)xe2x88x924 than (xc2x1)xe2x88x923, as observed. The results indicate that the importance of mesylate and triflate functionalities at the C-3 position on the biological activity of cephalosporins. This is in agreement with our hypothesis regarding their mode of action in biological systems (Scheme 1).
A carboxyl group at the C-4 position of cephalosporins 1 is essential for recognition by the target enzymes, such as penicillin binding proteins (PBPs) [Neu, H. C. Structure-Activity Relationships: Biological. In The Chemistry of xcex2-Lactams; Page, M. I., Ed.; Blackie Academic and Professional: New York, 1992; pp 101-128]. xcex2-lactams (xc2x1)xe2x88x923 and (xc2x1)xe2x88x924 are substrates of PBPs. On the other hand, their benzyl ester derivatives (xc2x1)xe2x88x9211 and (xc2x1)-12, lack of a carbonxyl group at the C-4 position, do not exhibit antibacterial activity (Table 1) although they both possess excellentxe2x80x94SO2Me and xe2x80x94SO2CF3 leaving groups. Thus, both the chemical reactivity and the recognition capability of a substrate by the target enzymes are essential for its biological activity, as expected.
Isodethiaazacephams bearing a good leaving group at the C-4 group position can undergo an enzyme-initiated 1,2-elimination as shown in Scheme 2. We found that mesylated xcex2-lactam (xc2x1)xe2x88x925 indeed exhibites profound antibacterial activity (Table 1).
It is apparent that the 7-phenylacetamido group in the xcex2-lactams (I) and (II) of the present invention may be replaced by various substituted acetamido groups used in the known antibacterial agents to further enhance the antibacterial activity thereof. Suitable substituted acetamido groups include (but limited to) phenoxyacetamido and the following side chains of cephotetan, cefotaxime, cefotoxin, moxalactam, cefazolin, cefazodone, cefatrizine, cefamandole, ceftiofur, cyclacillin and ampicillin: 
wherein Me represents methyl.